The antigen-specific cytotoxicity mediated by cytolytic T lymphocytes (CTL) is an important cellular mechanism of antiviral and antitumor immune responses. The ultimate goal of our studies is to reveal the cellular and molecular requirements for CTL effector functions and differentiation of immature CD4+8+ thymocytes into functional CD8+ CTL. We are currently focusing on the functional role of a novel set of cell surface proteins - the P2 (extracellular ATP) and P1 (adenosine) classes of purinergic receptors, which we propose to be intimately involved in CTL generation. According to our hypothesis the signaling through purinergic receptors may interfere with TCR-triggered activation pathways in differentiating thymocytes and may also be part of the apoptotic pathway leading to lymphocyte death. These studies may help us to understand the molecular mechanisms of immune response; additionally, they may provide novel targets for immuno-modulation. ATP0-triggered [Ca++]i increases in different subsets of thymocytes have been observed, providing evidence that thymocytes do indeed express functional purinergic receptors. A quantitative competitive RT-PCR method has been developed to study the expression of P2 receptors and used to show that apoptotic stimuli (steroids, ATP, adenosine) transiently upregulate expression of purinergic receptors. Signaling through purinergic receptors may have dramatic consequences in thymocyte development as evidenced by differential susceptibility of immature DP and SP thymocytes to apoptosis-inducing signals through P1 (adenosine) and P2 (ATP) purinergic receptors in both short-term (4-16 hrs) and long-term (5-7 days) fetal thymus organ culture assays. Signaling through P1 and P2 receptors has been shown to be able to interfere (antagonize or synergize) with TCR- and steroid-triggered apoptosis triggering in thymocytes. Inhibition of protein synthesis (in experiments modeling early apoptotic events in thymocytes) enhances the expression of P2 receptors. These results are compatible with the outcomes predicted by the purinergic receptors model of thymocyte differentiation. Experiments (targeted inactivation of purinergic receptors with antisense mRNA oligos, transfected constructs, and P2x gene knockout mice in vivo) are in progress to determine the contribution of individual P2 and P1 receptors to T cell functions and differentiation. These studies are expected to be greatly simplified by use of the in vitro differentiating, immortalized thymocyte clones that we established from p53 KO mice.